Field of the Invention
The present invention relates to a method which enables an individual's immune repertoire to be described, and thus certain pathological states to be detected and/or monitored.
An essential feature of the immune system is the capacity to recognize specifically a large number of antigens. In vertebrates, the T and B lymphocytes mainly execute this function of recognition, by means of at least three transmembrane molecular complexes; immunoglobulin for B cells, and the two T receptors for T lymphocytes: the .alpha..beta. receptor and the .gamma..delta. receptor, which thus define two subpopulations among the collective T lymphocytes (Wilson et al., 1988; Raulet, 1989).
To the tremendous variety of antigens to be recognized, there corresponds a very wide potential diversity of these three types of receptors. In effect, these three molecular complexes, of related structure, are composed of two (for each of the T receptors) or four (for immunoglobulins) peptide chains, the NH.sub.2 -terminal domains of which are highly variable. It is the existence of a strong interaction between a given antigenic determinant and the site consisting of these variable domains which constitutes the expression at molecular level of the phenomenon of recognition. Thus, the information contained in the genome of a mouse enables it to produce potentially at least 10.sup.11 immunoglobulins of different variable region, 10.sup.15 different .alpha..beta.T receptors and 10.sup.18 different .gamma..delta.T receptors.
The notion of repertoire thus emerges: the set of immunoglobulin variable regions present at a given instant in an organism constitutes the current repertoire of immunoglobulins. Similarly, the set of .gamma..delta.T-receptor variable regions possibly capable of being produced by a mouse genome constitutes the potential repertoire of .gamma..delta. lymphocytes.
The immune system hence contains, in fact, three complex repertoires, since the T-receptor repertoire must be subdivided into an .alpha..beta.T-receptor and a .gamma..delta.T-receptor repertoire.
The mechanisms which enable the huge diversity of T- and B-lymphocyte receptors to be produced are now well known (Tonegawa, 1983). The variable region of an immunoglobulin or of a T receptor is composed of the NH.sub.2 -terminal domains of two peptide chains. The genes coding for these two proteins are the result of somatic rearrangements which juxtapose a V segment, one or two D segments depending on the chains, and a J segment. The number of V, D and J segments available in the different loci provides a first source of diversity, termed combinatorial diversity (see Table 1). Furthermore, the lack of precision in the junctions between two of these segments (V-D, D-D, D-J or V-J junctions) introduces a second source of diversity, termed junctional diversity, since, on the one hand each of the two juxtaposed ends can have a few bases cleaved from it, and on the other hand some nucleotides can be inserted at the joining site. Lastly, in the case of genes coding for immunoglobulins, somatic mutations can take place in the second exon of the rearranged gene, thereby constituting a third source of diversity.
Depending on the type of receptor, .alpha..beta., .gamma..delta. or immunoglobulin, each of these three sources of diversity is a greater or lesser component of the total diversity. Thus, while the number of V, D and J segments available is smallest in the case of the .gamma..delta. receptor, the extent of the repertoire of this receptor remains potentially very large. This essentially stems from the fact that the gene coding for the .delta. chain can comprise zero, one or two D elements, which can, furthermore, be read in the three reading frames, whereas the V.sub.H and .beta. chains possess one and only one D element. In fact, the diversity of the .gamma..delta.-receptor repertoire may be described in the following manner: it appears that a small number of V and J segments is available, but the number of V segments of the .delta. chain is still not properly known, and there is a very wide potential junctional diversity which manifests itself in a large variation in the length of the second exon of the rearranged gene, especially in the case of the gene coding for the .delta. chain (Raulet, 1989).
The function of the .alpha..beta.T lymphocytes is relatively well known: they participate in cytolysis by killer cells, in reactions which regulate antibody synthesis and in inflammatory phenomena. The function of the .gamma..delta.T lymphocytes is still poorly understood: it is generally accepted that, apart from their probable role in the ontogeny of the immune system, the .gamma..delta.T cells participate in immune surveillance. As regards the various functions of antibodies, these are relatively well known and will not be restated here.
There is nothing at present capable of describing the collective antibodies and T receptors (i.e. the Ab and the TcR repertoires) expressed at a given moment in an individual. It represents a monumental task, since each of the repertoires probably contains millions of different molecules. Only a small number of reagents capable of specifically recognizing this or that element of such a repertoire is as yet available. It is, of course, possible to work more shrewdly by determining the sequence of a certain number of expressed genes. However, practical considerations make it scarcely conceivable to analyze routinely more than about ten or, perhaps, a hundred genes, and the operation is expensive and very lengthy. In short, the repertoires of antibodies and of T receptors are described at the present time only by means of a small number of parameters. Hence methods are not available which permit a rapid and effective analysis of the physiological an pathological situations associated with the statement of these repertoires. For example, it is clear that these repertoires vary during a voluntary immunization (vaccine), or during infection by pathogenic microorganisms, or during the progress of autoimmune pathologies. In the latter case, there are many reasons to believe that predisposition to these diseases mirrors a certain composition of the repertoires. It is hence very probable that a good method of analysis of the repertoires would have medical spin-offs, and could form the basis of techniques of medical analysis and of diagnosis.
A method which is now very widely used for the purpose of studying the diversity and distribution of the three repertoires, of immunoglobulins and .alpha..beta. and .gamma..delta. receptors, is that of amplification by PCR (polymerase chain reaction). This technique consists in amplifying genomic DNA or complementary DNA with a series of specific primer pairs (V,C) or (V,J) and, where appropriate, cloning and then sequencing the amplification products obtained (Takagaki et al., 1989); Asarnow et al., 1989). This powerful method is not without artifacts. Setting aside altogether the problems associated with quantification, it seems, for example, risky to deduce from two amplifications carried out with two different pairs of primers (V1, C) and (V2, C) for example, a preferential utilization of one segment relative to the other in the population under study (Rajasekar et al., 1990). In the current state of the art, it is difficult to determine by this method the degree of utilization of the different V segments. Moreover, cloning of the amplification products for the purpose of determining their sequence can also generate artifacts. For example, a certain proportion of these products are, in fact, heteroduplexes (if the amplified population is heterogeneous), for which it cannot be predicted how it will be "repaired" after transformation of the bacterium (Abastado et al., 1984, 1987).
A recent improvement is amplification according to the anchored PCR technique, which consists in carrying out the amplification of a heterogeneous population of complementary DNA using a single pair of primers, one hybridizing in the constant region C, the other with an identical sequence added at the 3' end of all the complementary DNA strands. It may hence be hoped that the amplification yield will not depend on the V segment used in the rearrangement.
Recently, a fairly sensitive method, enabling the degree of utilization of the different V segments in a population of heterogeneous transcripts to be evaluated, has been developed (Okada et Weissman, 1989; Singer et al., 1990). This accurate and reproducible method has the advantage of being conceptually simple, and hence of introducing little bias. However, it does not enable the V utilization and the J utilization to be defined simultaneously, nor does it enable it to be determined whether the transcript visualized is in frame.
Concomitantly with amplification from polyclonal populations, another widely used approach has consisted in constructing banks of hybridomas or of clonal T lines, and then characterizing at molecular level the antibodies or T receptors expressed. This method is obviously difficult to carry out on a large scale. It introduces biases which are difficult to evaluate during the steps of fusion or cloning, but is the only current technique that enables the sequence of both chains of a T receptor to be determined simultaneously, or a repertoire of a known specificity to be determined.
It should be noted that the methods mentioned above all characterize the repertoire from the messenger RNA and not from the protein, so that any post-transcriptional control is not known. The use of monoclonal antibodies and of flow cytofluorometry enables the repertoire to be analyzed at the level of the receptor itself, and gives simultaneously a large amount of information about the phenotype of the cells under study. However, this method is rather insensitive and, most particularly, does not enable the repertoire to be studied in detail since it is impossible, in the absence of reagents, to gain access to the junctional diversity of the repertoire. Furthermore, it requires the availability of a large battery of well-characterized monoclonal antibodies.